The objectives of this proposal are to investigate the molecular mechanism of the tRNA-guanine transglycolase (tgtase) reaction and the molecular interactions between the enzyme and both the nucleic acid substrate and the small molecule substrate. This enzyme is involved in posttranscriptional modification of tRNA and has been linked to differentiation and tumorigenesis. While this enzyme has been known for some time, no in vitro mechanistic investigations have been reported. A better understanding of the mechanism of action of tRNA-guanine transglycolase may provide insights into the biological role(s) of the queuine modification of tRNA. These objectives will be achieved by performing detailed, in vitro kinetic analyses of the Escherichia coli tgtase reaction using chemically-defined, homogeneous substrates. Analogues containing modifications of the structures of both the tRNA substrate and the small molecule substrate will be used to probe the mechanism of the tgtase reaction and to study the molecular determinants for both recognition and reactivity. An in vitro transcription system using the T7 RNA polymerase has been established to produce milligram quantities of tRNA-tyr (E. coli) and structural analogues. Structure-activity studies of the small molecule substrate will be performed on substrate and inhibitor analogues synthesized in collaboration with Professor L. B. Townsend (University of Michigan, College of Pharmacy). Preliminary studies have been performed using tgtase that has been isolated and purified as naturally expressed in E. coli. DNA amplification techniques have been used to subclone the tgt gene into an overexpressing vector. This will facilitate the production of sufficient quantities of enzyme enabling more detailed studies of the enzymic reaction and will allow for the production of mutant enzymes for future studies. Ultimately, this research will lay the groundwork for detailed investigations of the molecular interactions between the enzyme and both substrates. These investigations will help to elucidate the molecular mechanisms involved in protein-nucleic acid and protein-small molecule recognition and in enzymic catalysis.